Anti-angiogenic therapy is based on a simple, elegant theory: eradicate tumors by depriving them of their blood supply. An emerging concept in the field of matrix biology is that several heparan sulfate proteoglycans such as perlecan act not only as structural constituents during development and in tissue homeostasis, but also as key signaling molecules in tumor angiogenesis. We have discovered that the C-terminal fragment of perlecan protein core, named "endorepellin" to signify its anti-endothelial activity, is a powerful angiogenesis inhibitor. We have further discovered the functional receptor for endorepellin, namely the a2[unreadable]1 integrin, and shown that recombinant endorepellin blocks endothelial cell migration, collagen-induced capillary morphogenesis, and growth of blood vessels in Matrigel in vitro and in vivo. More recent results have demonstrated that endorepellin targets tumor vasculature in vivo and reduces the growth of orthotopic tumor xenografts and syngeneic tumors in mice. In spite of this accumulating evidence, the precise mode of action of endorepellin is not fully established. Our long-term objectives are to expand our initial observations and investigate in more depth the mechanism of action of endorepellin, identify novel genes affected by the action of this powerful protein, and systematically test its in vivo anti-angiogenic activity. Specifically, we plan to: [1] Decipher the precise mechanism of binding of endorepellin on the I domain of the a2[unreadable]1 integrin and its structural requirements, [2] Determine the precise mechanism of internalization, intracellular fate and downstream signaling of endorepellin in endothelial cells, and [3] Establish its in vivo anti-angiogenic activity by utilizing animal tumor models possessing or lacking the endorepellin functional receptor. Collectively, the expected results should generate a coherent and unified mode of action for endorepellin, identify new target proteins involved in its signaling pathway which could be potential novel targets for anti-angiogenesis therapy, and establish its in vivo role as an anti-angiogenic factor using unambiguous loss-of-function experiments through gene targeting. A better understanding of the biology of endorepellin would benefit research not only in cancer but also in other fields where angiogenesis plays a key role. The anti-angiogenic approach to controlling cancer can eventually succeed only if we deepen our understanding of angiogenesis and discover new compounds that modulate this key biological process. This proposal is focused on understanding the precise mechanism of action of a newly-discovered anti-angiogenic protein named endorepellin. Targeting the established tumor vasculature represents an alternate and potentially important therapeutic modality against cancer.